Proper folding to attain a defined three-dimensional structure is a prerequisite for the functionality of a protein. Improper folding that eventually leads to formation of protein aggregates is a hallmark of several neurodegenerative disorders. Loss of protein homeostasis triggered by cellular stress conditions is a major contributing factor for the formation of these toxic aggregates. A conserved class of proteins called chaperones and co-chaperones is implicated in maintaining the cellular protein homeostasis. Expanding the body of evidence highlights the role of chaperones as central mediators in the formation, de-aggregation and degradation of the aggregates. Altered expression and function of chaperones is associated with many neurodegenerative diseases including Parkinson’s disease. Several studies indicate that chaperones are at the center of the cause and effect cycle of this disease. An overview of the various chaperones that are associated with homeostasis of Parkinson’s disease-related proteins and their role in pathogenicity will be discussed in this review. Progressive loss of neurons is the most important characteristic of age-associated neurodegenerative diseases . A commoSNCA/PARK1), Parkin (PRKN/PARK2), PTEN-induced putative kinase 1 (PINK1/PARK6), leucine-rich repeat kinase 2 (LRRK2/PARK8), DJ-1 (PARK7), vacuolar protein sorting-associated protein 35 (VPS35/PARK17), ATP13A2 (PARK9) and glucocerebrosidase (GBA) are some of the extensively studied proteins for their association with PD .,70.69,70α-synuclein assemblies has been proposed and is most likely the case in young and healthy organisms [Selective degradation of ubiquitin-conjugated substrates via the 26S proteasome is called UPS . PD-relarganisms . As mentrganisms . Hsp70 erganisms . This surganisms . Anotherrganisms . CHIP, arganisms . CHIP-merganisms . Interesrganisms . The N-trganisms . The TPRrganisms . The TPRrganisms . In LBs rganisms . Overexprganisms . CHIP-merganisms . BAG1, arganisms . BAG1 inrganisms ,82. Howerganisms . Several DNAJ proteins are linked to PD, and a role for DNAJB6, a member of this family in the suppression of α-synuclein aggregation and proteasomal degradation, has been reported . KnockouMost of the ubiquitin-ligases are reported for polyubiquitinate α-synuclein; however, there are a few involved in the monoubiquitination of α-synuclein . Seven IStudies also revealed that increased levels of α-synuclein could inhibit proteasomal activity . OverexpCytosolic proteins with the specific amino acid sequence KFERQ are unfolded and transported into the lysosome for degradation via CMA. Hsp70, Hsc70 and other cooperating chaperones are required for protein degradation via CMA. CMA recognition motifs have been identified in α-synuclein, and further studies confirmed this to be an important mode of degradation of the protein ,94. AccuA recent study highlights the interplay between PD-associated proteins DJ-1 and α-synuclein . InhibitA53T transgenic mice. Overexpression of BAG3 in these cells resulted in enhanced degradation of α-synuclein via macroautophagy [Macroautophagy, commonly referred to as “autophagy”, is responsible for the degradation of larger protein aggregates. It is a nonselective process where the formation of de novo double membrane-vesicles (autophagosome) sequester intracellular components and traffic them to the lysosomes ,106. Atgutophagy . Inside the lysosome, the aspartyl protease cathepsin (CTSD) was reported as an important protease that degrades α-synuclein. Inactivation of CTSD in cells resulted in an increased level of α-synuclein . AccumulApart from the above prominent chaperone-assisted degradation mechanisms, a role for proteases such as calpains and neurosin in the cleavage of normal or aggregated forms of intracellular α-synuclein has been proposed . 2+ dysregulation, synaptic dysfunction, altered glutamatergic synaptic transmission and α3-Na+/K+-ATPase activity as a result of extracellular α-synuclein have been reported [α-synuclein is observed to be both intracellular and extracellular. Interestingly, larger aggregates are also reported to be transported to the extracellular space . Intracereported ,133. Relreported . Hence, reported . StrategSNCA have been identified that contribute to the development of PD. PRKN, PARK7, LRRK2, PINK1, POLG, ATP13A2, FBX07, GBA, PLA2G6, VPS35, DNAJC6, SYNJ1 and VPS13C have been reported with high confidence to be associated with PD [Several genes other than  with PD . In thisPRKN encodes a 465-amino-acid, 52-kDa multifaceted E3 ubiquitin ligase called Parkin (Park2). It has a ubiquitin-like domain on its amino terminal followed by a 60-amino-acid linker and four zinc finger domains. Like other E3 ligases, the last three zinc finger domains of Parkin contain two RING domains flanking a cysteine-rich domain called in between RING fingers (IBR). The physiological function of Parkin in the regulation of mitophagy and the degradation of proteins has been widely accepted  and sporadic PD [radic PD . Interacradic PD . In lineradic PD . The autradic PD . Kalia aradic PD . BAG5 inradic PD . PD-assoradic PD . Yang anradic PD . Parkin radic PD . The cytradic PD . Expressradic PD . HSPJ1 aradic PD . Interacradic PD . Interesradic PD . A role radic PD . Ubiquitradic PD .PINK1 encodes a putative protein kinase comprising of 581 amino acids and a calculated molecular mass of 66 kDa. Upon proteolytic cleavage, a 55-kDa mature isoform is generated [enerated . PINK1 ienerated . The phyenerated ). PINK1 enerated . Similarenerated  PINK1 inenerated . The mutenerated . Interacenerated . Loss ofenerated . A role enerated . BAG2 waenerated . TRAP1 . Howeve tissues . These m tissues . Autopho tissues . G2019S  tissues . Chapero tissues . Hsp90 s tissues . CHIP ub tissues . In acco tissues . Interac elegans . Inhibit elegans ,182. LRR elegans . However elegans . The bacterial DNAJ family and the homologous Hsp40 proteins function as co-chaperones for Hsp70. This family of proteins consists of one to four domains and share a consensus 70-amino-acid J domain . These pA role in clathrin dynamics and, therefore, in the pathogenesis of PD has been proposed for multiple DNAJC proteins listed above. However, further investigations are necessary to shed light on the mechanism of action of these proteins in this pathogenesis of PD .Research till now confirms that PD is not a single factor disease but has multiple factors associated to it and, at the same time, that the interactions between environmental factors and the genetic makeup of the person is very crucial. It is an age-associated neurodegenerative disorder and is majorly characterized by loss of proteostasis that eventually results in accumulation of toxic protein aggregates. Several proteins linked to the disease and the chaperones and associated co-chaperones involved in the proteostasis have been discussed in this manuscript. α-synuclein is the most important protein for which misfolding and aggregation into LBs is a characteristic feature of the disease. These protein aggregates hamper several essential cellular processes and subsequently affect neuronal viability. Treatment strategies targeting chaperones involved in PD that can restore the homeostasis of the target protein is also gravely explored. Development of patient-specific iPSC models of PD with the aid of novel genome editing tools has been instrumental in understanding the disease. However, to date, there is no cure to this disease and treatment given is only at alleviating the symptoms. As cellular structures are dynamic entities, a holistic approach at various factors in the pathogenesis is needed. An approach to look at both lipid and protein homeostasis together is essential to understand any interplay. Intracellular stress caused to organelles such as mitochondria and ER due to various aging-associated factors is also an important contributor to the disease. In this context, organelle interactions and association of the various proteins to different cellular structures may also shed more light on the molecular details. Posttranslational modifications of the PD-associated proteins and effect of these on protein–chaperone interactions and homeostasis of the proteins are also avenues that can shed light on the pathogenesis of the disease. The full functional relevance of several of these modifications and their interactions in both the physiological and pathological contexts is yet to be deciphered in detail.


BackgroundColorectal cancer (CRC) is one of the leading types of cancer worldwide and in Saudi Arabia. At the molecular level, CRC is very complicated and requires establishing comprehensive patient stratification models through identification of patients who will benefit or will not benefit from targeted therapy. We retrospectively investigated and analyzed the frequency of Kirsten-ras (K-ras) mutation and its correlation with patients’ characteristics as weel as its association with clinicopathological features  in patients with CRC.MethodsMedical records and paraffin-embedded tumor samples from 51 patients with histologically proven colorectal adenocarcinoma referred to Madinah center in Saudi Arabia were analyzed for the occurrence of rat sarcoma virus (RAS) mutations.ResultsRAS mutations occurred in 43% of the patients; 91% of these mutations were in K-ras. Seventy-five percent of these K-ras mutations were in codon 12, most commonly p.G12D. Codon 13 mutations occurred in 20% of tumors: all of these were p.G13D (100%). The percentage of K-ras mutations occurrence was higher in young patients (≤50) compared with the older patients (>50) . Similarly, the percentage of K-ras mutations occurrence was higher in the right-sided tumors compared with the left-sided tumors . Patients’ characteristics and clinicopathological features were not significantly associated with K-ras mutations.ConclusionsK-ras mutations are common among Saudi patients diagnosed with CRC in Madinah, especially pG12V and pG12D in codon 12. Further investigation would be required to establish correlation of K-ras mutations in larger cohorts. Colorectal cancer (CRC) is one of the leading types of cancer worldwide ,2. In SaKirsten-ras (K-ras) is an oncogene that is reported to be activated through mutations in 35% to 45% in patients with CRC . Almost In this study, we aimed to investigate retrospectively the frequency of K-ras mutation and analyze its correlation with patients’ characteristics and its association with clinicopathological features  in patients with CRC.Study populationA retrospective study was done at King Fahad Hospital, Madinah, Saudi Arabia. Histopathological and clinical data were obtained from the medical records of 51 patients with histologically proven colorectal adenocarcinoma. Tumor samples were obtained during surgery or biopsies carried out as part of routine care between 2016 and 2019. The histopathology reports were retrieved from the hospital’s laboratory information system and were analyzed for data pertaining to age, gender, site of tumor, histologic type, tumor grade, nodal status and stage. The medical imaging records provided information on metastatic site. The RAS mutation results for all the cases were known, testing was done at Genekor Labs, Greece. All aspects of the study were approved by the local ethics committee.K-ras and N-ras mutation testingDNA extraction was carried out from formalin fixed paraffin embedded tissue. 6µ thick sections were taken on positively charged slides from a tumor block selected by the primary reporting histopathologist. These were sent to a referral laboratory . Macrodissection of the tissue was performed on direction of the histopathologist at the referring laboratory by marking the area on the template slide. Tumor cell content (TCC) >75%. DNA was extracted from the sample under investigation .A targeted resequencing assay  was used for mutation detection in exons 2, 3 and 4 of the K-ras and N-ras genes. Sequencing was carried out using the Next Generation Sequencing Platform Ion Gene Studio S5 Prime System (Thermo Fisher Scientific). The kit was used to detect mutation in p.G12S, p.G12R, p.G12C, p.G12D, p.G12A, p.G12C on Codons 12 and 13 of exon 2. Exon 2 wild type cases were further analyzed for mutations in exons 3 & 4 of K-ras genes and exons 2, 3 & 4 of N-ras genes. The detection limit of the used method was 2%-5% of mutant allelic content, depending on the genomic region analyzed.Study designThe overall frequency of the K-ras and N-ras mutations including type of mutation and affected codon were tabulated for all the cases with respect to age, gender, tumor site, tumor histologic type, American Joint Committee on Cancer (AJCC) stage and site of metastases. SPSS software (v20) was used to perform the statistical analysis. P <0.05 was considered statistically significant.1) Characteristics of patientsFifty-one patients with CRC were evaluated. The mean age was 60.2 years . There was no difference in male to female ratio (i.e 1.04:1). In addition, all stages are included in the cohort. Most tumors were left sided (i.e 62.7%). Adenocarcinoma accounts for 94% of the tumors and the site of metastasis was mainly liver. All the other tumor characteristics are shown in Table 2) Characteristics of K-ras mutationsMutational analysis revealed RAS mutations in 43.1% 22/51) of CRC patients (Table 2/51 of C3) Correlations between K-ras status (mutation vs wild type) and clinicopathological featuresThe percentage of K-ras mutations occurrence was higher in young patients (i.e ≤50) compared with the older patients (>50) . Similarly, the percentage of K-ras mutations occurrence was higher in the right-sided tumors compared with the left-sided tumors  . We have found that 43% of the cohort carries RAS mutations with one-third of these mutations localized within codon 12 of K-ras. Characterizing the clinicopathological features of K-ras mutations in CRCs has carried increasing attention in recent years. One of the main reasons is due to the fact that CRC patients with K-ras mutations will not benefit from anti-EGFR antibodies .Percentage of K-ras mutations, which are known to work as an activated oncogene, occurs in 35%-45% of CRC worldwide ,11,16. ROne of the main objectives of this study is to look into K-ras status and its correlation with patients’ characteristics as well as its association with clinicopathological features. We have found that the percentage of K-ras mutations occurrence was higher in young patients (i.e ≤50) and in the right-sided tumors. However, we found no significant association between the overall frequency of K-ras mutations and patients’ characteristics and/or clinicopathological features. In literature, conflicting results were documented regarding the association between K-ras mutations and patients’ characteristics and/or clinicopathological features -21. For Delineating the pathogenetic importance of mutant K-ras in CRC is a successful step towards the control and management of this common type of cancer. In this study, 75% of K-ras mutations were found in codons 12 and 20% in codon 13. A similar observation was reported by others in which they found ~87% and ~14% of K-ras mutations in codons 12 and 13, respectively ,19,24. WIn this small retrospective study, we showed that the rate of K-ras mutation in Saudi patients with CRC is 40% which is in agreement with most studies worldwide. K-ras mutation status did not significantly correlate with patients’ characteristics and clinicopathological features. Lastly, our data has suggested that further investigation would be required to establish the pathogenetic importance of K-ras mutations in larger cohorts as well as to address the correlation and its prognostic value. Furthermore, longer follow up to address the survival are warranted.


Heliopsis longipes (Asteraceae) accumulates in root a C10 alkamide called affinin in its roots, but not in leaves. The closely related species Heliopsis annua does not produce alkamides. To elucidate the biosynthetic pathway of the alkamides acyl chain, a comparative global gene expression analysis contrasting roots and leaves of both species was performed.Alkamides are plant-specific bioactive molecules. They are low molecular weight N-substituted α-unsaturated acyl amides that display biological explicit activities in different organisms from bacteria, fungi, insects to mammals and plants. The acyl chain has been proposed to be biosynthesized from a fatty acid; however, this has not been demonstrated yet. H. longipes roots, but not in tissues and species that do not accumulate alkamides. The first domain searched was the Ketosynthase (KS) domain. The phylogenetic analysis using sequences of the KS domain of FAS and PKS from different organisms, revealed that KS domains of the differentially expressed transcripts in H. longipes roots and the KS domain found in transcripts of Echinacea purpurea, another alkamides producer species, were grouped together with a high bootstrap value of 100%, sharing great similarity. Among the annotated transcripts, we found some coding for the enzymatic domains KS, AT, ACP, DH, OR and TE, which presented higher expression in H. longipes roots than in leaves. The expression level of these genes was further evaluated by qRT-PCR. All unigenes tested showed higher expression in H. longipes roots than in any the other samples. Based on this and considering that the acyl chain of affinin presents unsaturated bonds at even C numbers, we propose a new putative biosynthesis pathway mediated by a four modules polyketide synthase (PKS).Transcriptomics analysis allowed to identify genes highly expressed in alk) previously thought to be absent from the plant kingdom.The global gene expression analysis led to the selection of a set of candidate genes involved in the biosynthesis of the acyl chain of affinin, suggesting that it may be performed by a non-iterative, partially reductive, four module type I PKS complex (PKS  E) or conjugated , either in the α-, intermediate or methyl-ω position, starting at an even C position. Alkamides are plant metabolites comprising a group close to 300 structures produced by a limited number of plant species belonging taxonomically to only eight families. A database of alkamides and other acyl-amides has been recently compiled  where alkamides have been detected in the whole plant. Several species containing high levels of alkamides are found in the Asteraceae, Piperaceae and Rutaceae; being more frequent in the Asteraceae family  bonds, when present, are in a position corresponding to that of the double bonds, most frequently occurring as acetylenic or methyl acetylenic in the ω-position. When more than one acetylenic bond is present, these are conjugated. In those alkamides where the acetylenic bond is at the ω-position, the acyl chain presents an odd number of carbons. In Heliopsis longipes, alkamides accumulate in the roots but are not present in the vegetative aerial tissue. In contrast, alkamides have not been detected in leaves or roots of the related species Heliopsis annua. The most abundant alkamide in H. longipes roots is N-isobutyl-2E,6Z,8E-decatrienamide, named affinin. It represents over 90% of the total alkamide content in well-developed roots. Other C10 acyl chain alkamides in this tissue are structurally related to affinin, such as the N-2-methylbutyl-2E,6Z,8E-decatrienamide and the bornyl ester of C10-2E,6Z,8E  by successive β-oxidation, dehydration, and isomerization (cis configuration (Z) and non-conjugated. The double bonds of fatty acids are incorporated by desaturases at a site different from that on which the acyl chain is synthesized and are strictly positioned in relation to the first desaturation 9Z, such as 12Z, 15Z, or 18Z (trans configuration (E) but may be in cis (Z) when these are conjugated, in contrast to those found in fatty acids. These facts, together, suggest that the biosynthesis of the acyl chain of alkamides could occur through an alternative pathway. Biochemical or genetic information to support the fatty acid or any alternative biosynthetic pathway of alkamides is still scarce, essentially because alkamides are produced in non-model plants for which the genomes have not yet been sequenced. Whole genome sequencing has been successfully used to elucidate biosynthetic pathways in microorganisms, such as bacteria. However, plant genomes are much larger than bacterial genomes, which increases the cost and time associated with sequencing, and plant genomes exhibit particularities such as alternative splicing that can give rise to different transcript versions of a single gene. Therefore, transcript profiling using high-throughput RNA sequencing (RNA-Seq) is a first-rate alternative to investigate the biosynthetic pathways of metabolites in plants without sequenced genomes . Voucher specimens were deposited at Instituto de Ecología, Centro Regional del Bajío. Herbarium IEB (Voucher 263787). Heliopsis annua was collected in October 2014 in the municipality of Vicente Guerrero, state of Durango, México . Voucher specimens were deposited at CIIDIR Durango Herbarium (Voucher id. D. Ramírez 5543). Fresh roots and leaves of both species were collected separately, washed with distilled water, immediately frozen in liquid nitrogen, and stored at −80 °C until processed for RNA extraction.Heliopsis species and processed separately as two replicates, following the protocol established by the PureLinkTM Micro-to-Midi Total RNA Purification System . The quality of the RNA samples was evaluated using the RNA 6000 Nanochip on the BioAnalyzer 2100 . Complementary DNA libraries were constructed with insert sizes from 339 to 697 base-pairs (bp) and sequenced using the Illumina HiSeq 2500 platform  with paired-end reads of 100 bp, at the Unidad de Servicios Genómicos of CINVESTAV, Irapuato, Guanajuato, México.Total RNA was extracted from roots and leaves of eight individual plants of each The resulting FASTQ files were pre-processed by removing adapter sequences and low-quality bases using the software package Trimmomatic-0.32, as previously described . Clean rwww.ncbi.nlm.nih.gov), Protein Family  database, which contains a large collection of protein families, each represented by multiple sequence alignments and hidden Markov models, and Swiss-Prot database (https://www.uniprot.org/), with an e-value cut-off of 1e−5. Gene Ontology (GO) analyses were further used to categorize the function of the unigenes with Blast2GO 5 (https://www.blast2go.com/). Using the basic local alignment search tool (BLAST), the Arabidopsis thaliana transcription factor (TF) database was queried to identify TFs among all unigenes (identity N80%) (The consensus contig sequence files were annotated against the non-redundant protein (NR) database from the National Center for Biotechnology Information  .p-value cut-off ≤0.05 [multiple test correction: false discovery rate (FDR)] and log2 fold change (FC) ≥2 were used to identify significant differences in transcript expression.To estimate the expression level of each transcript in the different tissue samples, high-quality reads from each sample were mapped onto the final transcriptome assembly using the RSEM software . Read coH. longipes and H. annua were obtained from the translated assembled RNA-Seq data. The KS domains were corroborated by BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and PFAM, a database of large collection with protein families (https://pfam.xfam.org). The domains compared were: the KS domains identified in H. annua roots (A1–A4); the KS domains upregulated in H. longipes roots (B1–B4); the KS domains related to FAS from H. annua (C1–C11); the KS related to FAS from H. longipes (D1–D11); the KS of the type III PKS from H. annua (E1–E4); and the KS of the type III PKS from H. longipes (F1–F4). These KS sequences were compared with KS domains of several microorganisms, plant and mammal species obtained from the NCBI database (www.ncbi.nlm.nih.gov) . Echinacea purpurea also belongs to tribe Heliantheae within the Asteraceae and produces alkamides that are structurally related to affinin (The amino acid sequences of the KS domains of nih.gov) . The KS  affinin . After s affinin . Phyloge affinin .www.primer3.org) and are shown in −ΔΔCT method (H. longipes roots vs. leaves and H. longipes roots vs. H. annua roots.Reverse Transcriptase quantitative polymerase chain reaction assays (RT-qPCR) were performed using the Applied Biosystems 7500 Fast Real-Time PCR system  with the Brilliant II SYBR Green RT-qPCR 1-Step Master Mix Kit . Gene-specific primers for the selected genes in the acyl chain biosynthesis pathway were designed using Primer3 . Roots and leaves of H. longipes generated 45,622,318 and 31,731,475 raw reads, respectively, whereas roots and leaves of H. annua generated 30,326,119 and 30,089,428 raw reads, respectively , followed by the MYB family (223 unigenes), and members of the GRAS family (80 unigenes). In H. annua, among the 938 unigenes related to TFs, the most abundant presented high homology to members of the LSD family (218 unigenes), followed by the WRKY family (162 unigenes), and the members of the HB family (68 unigenes). Finding MYB family members in H. longipes was interesting, since the MYB TFs include a conserved MYB DNA-binding domain that commonly binds to and regulates genes that code for enzymes involved in metabolic pathways , acyl transferase (AT), acyl carrier protein (ACP), oxide reductase (OR), dehydratase (DH) and thioesterase (TE) were selected  and the tissues that do non-produce these compounds .In order to confirm the differential expression of candidate genes in selected . Actin  and H. longipes (leaves), that were annotated as coding for a KS domain or KS domains related to FAS or type III PKS; and B for H. longipes roots upregulated transcripts. Other KS domains of FAS and PKS from mammals, microorganisms and other plants, obtained from NCBI listed in Echinacea purpurea were also included.The list of annotated transcripts differentially and not differentially expressed in H. longipes D1–D11 and H. annua C1–C11, are grouped in a clade together with the FAS from Arabidopsis thaliana KS with a high bootstrap value (98%). This result suggests that these transcripts, obtained from H. longipes and H. annua, encode enzymes with function similar to the KS domain of A. thaliana, which belongs to β-ketoacyl-[acyl-carrier-protein] synthases III (EC 2.3.1.180), enzymes that catalyze the chemical reaction of acetyl-CoA + malonyl-[acyl carrier protein] and produces acetoacetyl-[acyl carrier protein] + CoA + CO2. These enzymes belong to the family of transferases, and particularly to those transferring groups other than aminoacyl groups. They participate in fatty acid biosynthesis and are found in the dissociated or type II FAS biosynthesis system that occurs in plants and bacteria. The KS domains related to type III PKS are represented by the domain found in chalcone synthases (CHS), the enzymatic complexes mediating the synthesis of flavonoids in plants (H. longipes (F1–F4) are grouped together with a high bootstrap value of 100%. However, they are grouped in a clade far apart from the KS of type III PKS from H. annua E1–E4, which cluster in a clade together with the KS of type III PKS from Arabidopsis thaliana with a high bootstrap value of 98%. Therefore, these transcripts labeled as E1–E4 may share a similar function to the type III from A. thaliana. In addition, the translated transcripts of H. annua A1–A4 are close to the clade of H. annua E1–E4, translated transcripts coding for type III PKS KS domains or Chalcone synthase (CHS). As for the FAS-related sequences, the transcripts coding for these domains are present in both species, which could suggest that they may not be involved in the biosynthesis of the acyl chain of alkamides.The results of the maximum likelihood phylogenetic analysis based on 89 KS sequences are presented in n plants . The nonH. longipes roots, the species and tissue that produces alkamides, grouped with a high bootstrap value of 100% with KS (3-oxoacyl-[acyl-carrier-protein] synthase) of Echinacea purpurea roots. This group is exclusively formed by translated transcripts of E. purpurea and upregulated in H. longipes roots. This association suggest that the KS domains in both species probably have the same function and could be related to alkamide acyl chain biosynthesis, due to their functional nature. Close to this KS clade, KS domains from other plant species include Helianthus annus and Cynara cardunculus. This clade is not far from KS domains from the Chlamydiae phylum microorganism PKS specifically with the two orders: Chlamydiales and Parachlamydiales; and a single class Chlamydiales. These are obligate intracellular Gram-negative bacteria  The first KS domain, associated with plant FAS, includes general H. longipes, H. annua, and other plant transcripts; (2) A second domain type found exclusively in H. longipes root upregulated transcripts and E. purpurea. This KS domain, present in both species, is possibly related to the biosynthesis of the acyl chain of alkamides in H. longipes and E. purpurea. Interestingly, they were grouped in a clade far from FAS and PKS III KS reported in plants, but closer to microorganism PKS  The fourth includes two clades of H. annua KS domains related to type III PKS.In summary, the phylogenetic analysis revealed that the KS domains found in the annotated transcriptomes of the two species of the nism PKS ; (3) TheH. longipes alkamides is proposed. The available information about the different domain activities in published PKS systems, allowed the assembly of a model of a biosynthetic pathway that could produce the C10-2E,6Z,8E acyl chain of affinin. This model incorporates the KS, AT, ACP, OR (including the keto reductase (KR) and ER), DH and TE domains previously found. The model proposes a four-module PKS system where the starting precursor molecule is acyl-CoA, and the extender unit is malonyl-ACP  enzymes in a modular manner. As observed in polyketides biosynthesis from bacteria, fungi and symbiotic marine microorganisms such as in sponges , where uonyl-ACP .GO:0016740) domain accepts and loads the extender unit onto the ACP (GO:0016740) and produces malonyl-ACP. Then, the KS (GO:0016740) domain catalyzes a decarboxylative Claisen condensation with the acyl-CoA and the resulting product is a four carbon (C4) acyl chain with α- and β-keto groups. The KR, including OR (GO:0016491) and DH (GO:0016740) domains, sequentially reduce the β-keto group to a β-hydroxyl group and an α-E double bond, respectively. The α-unsaturated acyl chain is transferred by the AT (GO:0016740) to the second module (Aff-2) and is elongated by the ACP (GO:0016740) with another malonyl-ACP producing a C6-acyl chain. The same enzymatic reactions on the β-keto group as in the first module occur in module 2. The product is a C6-2Z,4E-dien-acyl chain, which is transferred to the module 3 (Aff-3) and condensed with malonyl-ACP to produce a C8-acyl chain with α- and β-keto groups. In this module, which consist of includes the KR including OR (GO:0016491) and DH (GO:0016740) domains, the β-keto group is reduced to a β-hydroxyl group and dehydrated to an α-E double bond. The ER (GO:0016491) enzymatic domain, present only in this module, reduces the double bond. In module 4 (Aff-4), the acyl chain C8-4Z,6E is once more condensed with malonyl-ACP to produce an C10-acyl chain with two keto α- and β- groups. A KR, including OR (GO:0016491) and DH (GO:0016740) domains, in this module reduces the β-keto group to a β-hydroxyl group, and the DH domains to an α-E double bond. The absence of an ER domain in this last module 4 results in the transference of the final C10-2E,6Z,8E-chain to the TE (GO:0016744) domain present at the end of this four module PKS. The proposed sequence of acyl chain biosynthesis, as evidenced by the differential expression supporting a four-modules multi-enzymatic complex, is as follows: in the first module (Aff-1), the AT  that act at the end of the FAS steps, or complexes such as PKS. It is responsible for the condensation of two substrates to generate or elongate an acyl chain, depending on where the enzyme that contains it is present. It can be assumed that such a domain would be participating in the initial steps of the biosynthesis of the acyl chain of alkamides. Indeed, transcripts encoding the KS domain were found among the differentially expressed set. The phylogenetic analysis  revealedn plants .Surprisingly, in the phylogenetic analysis comparing KS domains of FAS, PKS, and the putative alkamide acyl chain biosynthetic genes, KS domains of the latter were found in a clade close to PKS from microorganisms. Thus, PKS-related, specific genes, appear to be conserved within the alkamide producer species, suggesting duplications that led to the evolution of novel functions. A possibility is that these plants acquired these genes by horizontal transfer, as has been suggested for other PKS gene families  or alterH. longipes. However, since RNA-Seq analysis was performed using polyadenylated RNA, it is unlikely that prokaryotic RNA was sequenced, though fungal transcripts would not have been filtered out. Nevertheless, we would expect fungal transcripts to be present at much lower levels than plant transcripts. Moreover, putative endophytes that are exclusively present in H. longipes roots have not been observed in the in vitro root culture in the lab to date.Because of the clustering of KS domains coded by upregulated transcripts with KS domains found in type I PKS systems, found in certain microorganisms, we sought whether other similar domains present in those systems were also found coded by other upregulated transcripts. These results suggest alkamide synthesis is likely carried out by enzymes that are similar to type I PKS. These findings are important, as the differentiation of PKS and FAS will facilitate approaches investigating alkamides biosynthesis pathways in other plant species. They are also interesting as this type of systems were previously thought to be absent from plant species, and mostly present in microorganisms. One possibility is, of course, that these enzymes are expressed by endophytes that are exclusively present in the root tissues of To propose the novel alkamides biosynthetic pathway, we considered the chemical structure of the affinin acyl chain, and the characteristics of the known products of FAS or PKS. Fatty acids derived from FASs comprise completely reduced acyl chains, and the polyketides derived from PKSs include partially unsaturated acyl chains. In the polyunsaturated fatty acids (PUFAs), the double bonds are incorporated by fatty acid desaturases (FADs), iron-dependent metalloenzymes, on the fatty acids released from the FAS enzymatic complex . In contH. longipes roots through a pathway that is similar to the polyketide biosynthesis pathway. This model is further referred as “PKS alk”. The proposed PKS alk  would work in a modular fashion as in the known type I PKSs  module. However, in the Aff-2(γ) module, the DH would generate a cis double bond during the dehydration of an L-β-hydroxy intermediate. This mechanism, by means of which the DH generates cis double bonds when a trans double bound is inserted in the previous module during the synthesis of an acyl chain, has been observed in several PKS type I enzymes in microorganisms  and Ctenium aromaticum (Poaceae).The “PKS H. longipes and H. annua is described. The comparative transcriptome analysis performed for the two Heliopsis species led to the selection of a set of candidate genes putatively involved in the affinin acyl chain biosynthesis, allowing to propose a model for alkamide acyl chain biosynthesis by a PKS complex, here named PKS alk. This complex would be partially reductive and related to type I PKSs, and present in plants that produce alkamides. These findings provide a framework for molecular and biochemical studies to confirm the activity of the proposed pathway. Further studies can also help to better understand the underlying regulatory mechanisms involved and the physiological role of these multifunctional molecules in plants.In the present study, the first transcriptomic high-throughput sequencing of the Mexican endemic plants 10.7717/peerj.10074/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.10074/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.10074/supp-3Supplemental Information 3H. longipes roots and a relevant putative function according to the annotation, and the Locus, % identity, e-value, score and information about the alignments to Arabidopsis (green section) and other plant species (blue section) is presented, for the proposed multi-enzymatic complex PKS alk for the synthesis of Affinin.The table includes the Enzymes name, Unigenes, Sequences and GO that presented increased accumulation in Click here for additional data file.10.7717/peerj.10074/supp-4Supplemental Information 4This data was used to obtain Figure 5: Relative expression of nine candidate genes involved in the putative alkamide acyl-chain biosynthetic pathway.Click here for additional data file.


Scientific Reports 10.1038/s41598-017-10991-x, published online 06 September 2017Retraction of: The Editors have retracted this Article.An institutional investigation found evidence that the images used for Figures 3C, 3D, 3F, 5D and 6E, originated from a different set of experiments to those listed. The Editors therefore no longer have confidence in the integrity of the data presented.Authors Jaewang Lee and Daiha Shin agree to this retraction. Authors Eun Hye Kim and Jong-Lyel Roh have not responded to any correspondence from the Editors about this retraction.


These forms spectroscopically differ from that observed previously in oxidative redding (λabs/λem = 575/607 nm). We also report on a new green-emitting state (λabs/λem = 405/525 nm), which is formed upon photoconversion under the low-oxygen conditions. Based on the spectral properties of these forms, their light-independent time evolution, and the high-level computational studies, we provide a structural basis for various photoproducts. Under the low-oxygen conditions, the neutral quinoid-like structure formed via a two-electron oxidation process is found to be a key intermediate and a most likely candidate for the novel green-emitting state of the chromophore. The observed large Stokes shift is traced to the formation of the zwitterionic form of the chromophore in the excited state. Subsequently, this form undergoes two types of cyclization reactions, resulting in the formation of either the orange-emitting state (λabs/λem = 490/565 nm) or the red-emitting form (λabs/λem = 535/600 nm). The T65G mutant lacks one of the proposed cyclization pathways and, indeed, the photoconverted T65G EGFP exhibits a single orange-emitting state. In oxidative redding, the red-emitting state resembles the structure of the chromophore from asFP595 (λabs/λem = 572/595 nm), which is directly formed upon two-electron oxidation and deprotonation bypassing the formation of the quinoid-like structure. Our results disclose a general “oxidative” mechanism of various green-to-red photoconversions of EGFP, providing a link between oxidative redding and the photoconversion under low-oxygen conditions.Here we dissect the phenomena of oxidative and reductive green-to-red photoconversion of the Green Fluorescent Protein. We characterize distinct orange- and red-emitting forms (λ The original observations of photoinduced red-shift in the fluorescence emission of the green fluorescent protein in bacteria  under loin vivo .The fluorescent protein denoted as EGFP in this work has the following amino acid sequence:>EGFPMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT YGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMK QHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTL VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKN GIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH YLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKThe protein denoted as EGFP-T65G carries X = Gly instead of X = Thr in the chromophore-forming tripeptide (-X-Y-G-).Escherichia coli strain . The single bacterial colony was inoculated into 200 ml of LB medium with IPTG and ampicillin . The flasks were then incubated at 37°C with vigorous shaking (250 rpm). After 12 h 100 ml LB media with 10 mM IPTG and 200 mg/ml ampicillin was added in the culture and grown for 6–8 h.Fluorescent proteins were expressed in XL1 Blue g, 10 min, 4°C) and incubated with TALON Metal Affinity Resin (Takara Bio Europe S.A.S.) for 15 min. The resin was washed with PBS with 10 volumes of the column. The protein was eluted with PBS containing 100 mM EDTA (pH 7.4). Finally, all purified samples were concentrated and equilibrated in PBS (pH 7.4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis was performed in denaturing conditions according to standard protocols in 12.5% polyacrylamide gel.The culture was centrifuged, the supernatant was discarded, the pellet was resuspended in 5 ml of phosphate buffer (pH 7.4), the suspension was lysed in Sonics Vibra Cell sonicator . The cell lysates were cleared by centrifugation (16000 2) on the cuvette, and the entire apparatus was covered in light-reflective aluminum foil. We assessed photoconversion of fluorescent proteins in two major experimental conditions – those in favor of oxidative redding, containing strong oxidants described in the original paper (The cuvette with a 2 μM of fluorescent protein in 1 ml PBS solution was illuminated for 1 (for oxidative redding) or 5 (for reductive redding) minutes with focused blue light from seven ultra-bright light emitting diodes . The light was focused . Sodium dithionite reacts with the dissolved oxygen, therefore serving as both reductant and an oxygen scavenger. Alternatively, we used slow bubbling (for ∼15 min) of oxygen-free argon through the buffer to remove dissolved oxygen. In this case, active reductant was produced in the reaction of the photoreduction of FAD in the presence of EDTA 6].For oxidative photoconversion, we followed the protocol described previously . In the For the measurement of pH dependence of the fluorescence spectra, we used 50 mM buffers in the pH range from 3.5 to 11 . An aliquot of protein sample after photoconversion was diluted in the corresponding buffer solution and immediately placed in the spectrophotometer for measurements.SDS-PAGE analysis was performed using a 12% polyacrylamide gel. A 25 μg aliquot of protein was added to the loading buffer mix and adjusted to a total of 50 μl. The solutions were mixed well and heated to 100°C for 5 min in a PCR machine. 5 μg of protein (10 μl) was loaded to each lane of the gel. Electrophoresis was performed in the SDS-Tris-glycine buffer. The voltage for the concentrating gel was set to 100 V, and to 200 V for the separating gel. The gel was stained with Coomassie dye blue diamond G-250.Varian Cary Eclipse Fluorescence spectrophotometer was used to measure excitation and emission spectra. The absorbance spectra were acquired with Cary 100 UV/VIS spectrophotometer.Fluorescence-decay kinetics was collected by a time- and wavelength-correlated single-photon counting setup . The temperature of the sample was stabilized at 21°C by a Peltier-controlled cuvette holder Qpod 2e  with a magnetic stirrer. 2 min irradiation of the sample with focused light from 200 mW royal blue  LED (Thorlabs) was used for photoconversion. Excitation of EGFP was performed with ps-pulsed lasers at either 405 or 510 nm , driven at a repetition rate of up to 50 MHz. Longpass filter 510LP (Thorlabs) was used to filter out excitation light. A monochromator additionally filtered the emission  set to either 610 or 525 nm (21 nm FWHM). Finally, the emission was collected with a photon-counting detector  with a low dark count rate (∼10 counts per second). SPCImage  software package was used for approximation of fluorescence decay curves by a sum of exponential decay functions.Atomistic models of various forms of the S65T-GFP protein were constructed based on the X-ray data [PDB ID: 1EMA ]. The E2The final molecular dynamics (MD) structure was reduced for the subsequent quantum mechanical/molecular mechanical (method) (QM/MM) calculations by removing outer water molecules located farther than 2.9 Å from the protein. The total number of atoms in the initial green form of the S65T GFP protein was equal to 5410. The quantum mechanical (QM) part (154 atoms) included the chromophore and all nearby amino acid residues . Ground-state equilibrium geometry parameters were obtained at the PBE0/(aug)-cc-pVDZ//CHARMM level of theory using a mechanical embedding QM/MM scheme. All residues within 7 Å from the QM region were optimized, whereas the remaining MM residues were held fixed during the geometry optimization. QM/MM partial Hessian evaluation and vibrational analysis were performed to ensure that located stationary points were true minima. The details of the computational approach can be found elsewhere .Vertical excitation energies (VEE) were calculated using extended multiconfiguration quasi-degenerate perturbation theory XMCQDPT2  with modin vitro green-to-red photoconversion of EGFP with lowered oxygen. We followed experimental design similar to one in the report by 2S2O4) in the sample, without additional steps for oxygen removal due to intrinsic oxygen-scavenging activity of sodium dithionite , we observed a distinct peak with green emission peaked at 525 nm, red-shifted in comparison with the emission spectra of EGFP . ApparenWe followed the evolution of the emission of all three distinct spectral forms associated with the photoconversion of EGFP in low-oxygen conditions . The fluWe estimated the apparent pKa of green-emitting form as ∼6.5 . We measBackbone fragmentation is observed in SDS-PAGE of protein samples after photoconversion.Fluorescent proteins harboring labile bonds within the protein backbone (such as acylimine) are typically unstable in conditions of SDS-PAGE, as was observed for DsRed-like chromophores . Therefoin vitro in the literature, we performed time-resolved measurements. Two excitation/detection settings were used: 405/535 and 510/610 nm . We estimated the lifetime of red-emitting form arising in the course of oxidative redding of EGFP as ∼0.6 ns.Similarly, we measured the fluorescence lifetime of the photoproducts of EGFP photoconversion in the presence of sodium dithionite. The mean lifetime of the red emission upon 510 nm pulsed laser excitation was ∼2.1 ns. We exploited the light-independent increase of the fraction of the 565 nm-emitting form to unmix the observed lifetime to two distinct components: ∼1.4 and ∼2.7 ns, the amplitude of the component with the lifetime of 1.4 ns was rising in agreement with the increase of the 565 nm emission in the steady-state fluorescence emission spectra. Therefore we attributed the 1.4 ns component to the fluorescence lifetime of 565-nm emitting form and the 2.7 ns component to the form with an emission maximum at 600 nm. We further estimated the mean fluorescence lifetime of green-emitting form as ∼2.6 ns.The first position in the conservative chromophore-forming triade (-X-Y-G-) is typically restricted to a limited set of amino acids in orange-emitting fluorescent proteins, namely Thr, Lys, Ser, or Cys . We therab initio calculations have been used to characterize various spectral forms of the chromophore inside the S65T GFP protein and to aid the interpretation of the experimental results. The green form has been obtained previously , 554 nm (0.02), and 518 nm (0.2). The anionic chromophore in the triplet state most strongly absorbs at around 900 and 500 nm, which does not exclude its formation during the photoconversion, e.g., ; howeverThe green-emitting quinoid-like structure, if stabilized, can also undergo subsequent cyclization reactions, yielding, for example, the mOrange-like chromophore . AlternaThe calculated VEE of the mOrange-like chromophore is 535 nm with an oscillator strength of 0.98. This value is consistent with the experimental absorption maximum of the red-emitting form observed in the photoconversion under the low-oxygen conditions . According to the calculations, the first bright transition of the bicyclic structure is at 480 nm with an oscillator strength of 0.53. This corresponds to the absorption maximum of the orange-emitting form observed experimentally at around 480–500 nm. In this case, the large Stokes shift  can also be traced to the quinoid-like structure of the bicyclic chromophore in the ground state and to the formation of the predominant zwitterionic form in the excited state.As a result, the red- and orange-emitting forms observed upon photoconversion under the low-oxygen conditions can be attributed to the anionic cyclic and neutral bicyclic structures of the oxidized GFP chromophore, respectively. We note that the bicyclic structure can further be hydrolyzed, which is accompanied by cleavage of the peptide bond. Upon deprotonation of the hydrolyzed structure, the anionic far-red absorbing chromophore is formed with the calculated VEE of 630 nm. This far-red absorbing and emitting chromophore has not been identified experimentally, indicating that the cleavage does not happen within the timeframe of the experiment.In the current work, we have established conditions for efficient green-to-red photoconversion of EGFP in the absence of molecular oxygen, in agreement with the original report by Another type of green-to-red photoconversion is known for fluorescent proteins sharing His-Tyr-Gly chromophore-forming triad, first discovered in the Kaede protein from a stony coral . This tyWhile the experimental conditions used for the green-to-red photoconversion in this study may be considered “reductive,” it is hard to imagine a red-shifted version of the GFP chromophore which is not formed as a result of some type of oxidation. Moreover, the computational studies suggest that side chains of certain amino acid residues in the immediate vicinity of the chromophore may serve as primary electron acceptors in photoinduced electron transfer . Overallabs/λem = 405/525 nm, the orange-emitting form with λabs/λem = 490/565 nm, and the red-emitting form with λabs/λem = 535/600 nm. The red-emitting form in oxidative redding is characterized by λabs/λem = 575/607 nm (5 (406 nm) to the green-emitting state, structure 6 (480 nm) to the orange-emitting state, and structure 7 (535 nm) to the red-emitting chromophore detected upon photoconversion under the low-oxygen conditions. Structure 3 (565 nm) and, in particular, structure 4 (580 nm) can be assigned to the photoproduct in oxidative redding. Based on these results, we propose a general mechanism for various green-to-red photoconversions of EGFP.Experimentally, three emitting states have been identified upon the low-oxygen photoconversion of EGFP in the present work. These species have distinct spectroscopic properties: the green-emitting form with λ5/607 nm . Our cal5/607 nm : structu5). Its light-independent interconversion leads to the orange-emitting (6) as well as red-emitting (7) chromophores. These structures are formed following two different types of cyclization reactions, which may compete with each other. The delayed appearance of the 565 nm emitting form can be explained by a two-step process, where cyclization is followed by dehydration. At the same time, the red-emitting form appears promptly following the mOrange-like cyclization of the chromophore  has not been previously reported. Yet, the prominent rise and fall of its emission in a light-independent manner, accompanied by a subsequent rise of orange emission at 565 nm, suggests an important role of the 525-nm emitting form as an intermediate in conversion to the final photoproducts. Our computational results reveal the neutral quinoid-like form of the chromophore as a most likely candidate for this unusual green-emitting form  can be reached via photoinduced electron transfer (Overall, the proposed mechanism  is in gotransfer  or indirtransfer  of the ctransfer . However5 in To conclude, we offer a solution to the long-standing enigma on the appearance of the red-emitting forms of the GFP chromophore upon photoconversion in the presence of reducing agents. We show that these species are different from those previously detected in oxidative redding. Based on our combined experimental and computational studies, we clearly distinguish between various forms and assign them to particular structures of the emitting species. The stabilization/destabilization of the newly discovered green-emitting chromophore (structure The datasets generated for this study are available on request to the corresponding author.AM and KL devised the project. DG, AMB, and AM performed photoconversion experiments and steady-state spectroscopy. AM, KL, AMB, MB, and AVB analyzed the data. EM performed time-resolved fluorescence spectroscopy and analysis. AK, EP, and AVB performed computational studies. AM, MB, AK, and AVB devised the proposed mechanism of the photoconversion. AM and AVB wrote the manuscript. All the authors exchanged comments during the manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To determine whether artificial intelligence (AI) processed PET/CT images of reduced by one-third of 18-F-FDG activity compared to the standard injected dose, were non-inferior to native scans and if so to assess the potential impact of commercialization.SubtlePET™ AI was introduced in a PET/CT center in Italy. Eligible patients referred for 18F-FDG PET/CT were prospectively enrolled. Administered 18F-FDG was reduced to two-thirds of standard dose. Patients underwent one low-dose CT and two sequential PET scans; “PET-processed” with reduced dose and standard acquisition time, and “PET-native” with an elapsed time to simulate standard acquisition time and dose. PET-processed images were reconstructed using SubtlePET™. PET-native images were defined as the standard of reference. The datasets were anonymized and independently evaluated in random order by four blinded readers. The evaluation included subjective image quality (IQ) assessment, lesion detectability, and assessment of business benefits.p=0.237) for all scanner models, with “almost perfect” inter-reader agreement. There was no significant difference between datasets in lesions’ detectability, target lesion mean SUVmax value, and liver mean SUVmean value . No false-positive lesions were reported in PET-processed examinations. Agreed SubtlePET™ price per examination was 15-20% of FDG savings.From February to April 2020, 61 patients were prospectively enrolled. Subjective IQ was not significantly different between datasets (4.62±0.23, This is the first real-world study to demonstrate the non-inferiority of AI processed 18F-FDG PET/CT examinations obtained with 66% standard dose and a methodology to define the AI solution price.The online version contains supplementary material available at 10.1186/s40658-021-00374-7. Positron emission computed tomography/computed tomography (PET/CT) is widely used in various clinical applications, including the investigation of oncological and neurological disorders . The evoRecently, reconstruction algorithms have been developed to improve the quality of images acquired with reduced administered radiotracer. However, these procedures are complicated, time-consuming and do not produce satisfactory outcomes when the injected activity is significantly lower compared to the standard dose suggested by the procedure guidelines . MachineThe value proposition of artificial intelligence (AI) in healthcare has been well described ; howeverTM was trained on hundreds of multi-vendor PET and PET-CT datasets featuring a range of image quality. Deep learning processing time per series is typically between 1-2 min. SubtlePET™ software uses a convolution neural network-based algorithm to reduce noise and improve image quality of fluorodeoxyglucose (FDG) and amyloid PET and PET/CT images [According to the framework, the SubtlePET™ AI solution  was selected to be assessed in a single, European Association of Nuclear Medicine (EANM) Research Ltd. (EARL)  accreditT images , 13. EvePatients referred for 18F-FDG PET/CT during diagnostic work-up for oncological disease were screened for prospective enrollment. Inclusion criteria were (a) age >18 years; (b) FDG-avid malignancy; (c) glycemia <180 mg/dL; (d) adequate physical condition to allow them to remain still for approximately 40 min, for two consecutive PET scans. Claustrophobic patients were excluded. Patients meeting these criteria were approached to participate in the study. According to GDPR and institutional procedures related to the information provided to patients for the examination process, all the patients signed an informed consent form prior to any study procedures.PET images were acquired with three different 3D PET/CT scanners  from the same manufacturer  without time of flight (TOF) technology. 18F-FDG was provided by Advanced Accelerator Applications pharmaceuticals  in compliance with Good Manufacturing Practice (GMP) and in accordance with EANM procedure guidelines .For the purposes of this study, FDG doses were reduced by one-third compared to the standard injected dose to a patient with the same body weight, according to institutional procedure guidelines. All doses were injected via peripheral venous catheter.During the same day, patients underwent two sequential PET scans in continuous-bed-mode: a reduced dose acquisition scan (PET-processed) and a reference acquisition scan (PET-native). The PET-processed scan was acquired first at 60 min post-injection from skull base to mid-thigh. To simulate normal acquisition time and reduced injected dose, PET images for scanners 1 and 2 were acquired at 2.5 min per bed-position, while for scanner 3, images were acquired at 1.5 min per-bed position, in accordance with institutional procedure guidelines. Following the PET-processed scan, the PET-native scan was acquired for the same region without moving the patient. The PET-native images were acquired with an elapsed time, increasing the minutes per bed-position, to simulate normal acquisition time and standard injected dose. To define the PET emission acquisition time that simulated a full dose examination, a phantom study was performed on each PET/CT scanner using cancer imaging conditions, applying the following equation: standard time acquisition per bed × exp(900×λ) × 1.25 (s).Patients underwent one low-dose CT prior to PET-processed acquisition, for attenuation correction and anatomical correlation of PET findings. Emission data was corrected for randoms, dead time, scatter, and attenuation and was reconstructed iteratively by an ordered-subsets expectation maximization (OSEM) algorithm. Three different PET/CT scanners were utilized: Discovery ST-4, Discovery ST-16, and Discovery IQ. The Discovery-ST  combines a helical multi-slice CT scanner (respectively 4 and 16 slices) and a BGO block detector PET tomograph. The Discovery ST has good overall performances in 3D acquisition mode, with balanced performance of the several physical parameters influencing the final image quality. In particular, high sensitivity results in low statistical noise and the consequent recovery of spatial resolution in clinical studies. Furthermore, the good NEC response allows clinical studies to be optimized in terms of the signal to noise ratio. The Discovery-IQ combines a BGO-based PET tomograph with a 16-slice CT scanner; the software includes the algorithm VUE-point HD (VPHD). The Discovery-IQ PET/CT scanner with 5-ring detector blocks has the highest overall performance of the Discovery BGO-based scanners, with further improvement in sensitivity and counting rate performance. According to institutional processes, images were reviewed for artifacts by the technologist before the patient was discharged. Upon confirmation, PET-processed acquisitions were sent by the radiographer from the modality to the Subtle server (SubtleEdge) for processing. Incoming images were automatically anonymized and quality controlled (QC) according to the SubtlePET™ process. Images that passed QC were processed and were sent automatically to the Picture Archiving and Communication System (PACS) in an average time of 10 min.The PET-native images were defined as the standard of reference and were reviewed by two independent physicians who had access to all the clinical, imaging, and reconstruction data, to reach to consensus report that was delivered to the patient within 24 h.The PET-processed and PET-native datasets were anonymized, separated, and randomized allowing independent assessment of each dataset over a 4-week period, by four blinded board-certified nuclear medicine physicians, with more than 5 years’ experience . Each reviewer assessed all datasets. They were blinded regarding image acquisition, reconstruction technique, and clinical information. 18F-FDG PET/CT images were reported according to EANM procedure guidelines [For image quality, the PET datasets were rated on a 5-point scale  with scores 4 and 5 considered adequate to provide diagnostic confidence.Furthermore, each reviewer had to give their opinion as the whether they were reviewing the PET-processed or the PET-native dataset or if this was indeterminate.max of the largest lesion and the SUVmean of the liver were measured. SUV was defined as activity concentration (Bq/mL) divided by injected activity (Bq) normalized to body weight. The highest voxel value (SUVmax) and the mean voxel value (SUVmean) were obtained in a volume of interest (VOI) covering the entire tumor as defined by each reviewer. Considering that all PET-native images were acquired after the PET-processed images, a correction factor for the SUV values was calculated according to Appendix Lastly, the detectability of all lesions was evaluated in a per-lesion analysis. In patients with ten lesions or fewer, all lesions were assessed by the reviewers, while in patients with more than ten lesions, those ten with the highest standard uptake values (SUV) max were included in the analysis. In the two datasets, the SUVOnce the independent analysis of the native and processed datasets was complete, they were logged and unblinded. The whole dataset was scrutinized to determine whether the processed scans were non-inferior to the native scans. Quantitative assessment was of lesion detectability and SUV levels; qualitative assessment on subjective image quality. Inter-observer variability for image quality assessment was performed. Differences in results between PET/CT scanner models were also assessed.p values were obtained by the two-sided exact method at the conventional 5% significance level. Data were analyzed with R 3.6.1 . Once the analysis of the outcomes from the clinical evaluation was completed, an assessment of the business benefits was performed. The potential net savings from the use of SubtlePET™ were calculated using data from the whole PET/CT network, not just the single center, assuming replicability of results. A percentage of these savings was then agreed as a fair price for the AI solution.Descriptive statistics for categorical variables were presented as relative/absolute frequencies, while those for continuous ones as the median (range). The inferential analyses for categorical and continuous variables were performed by the Fisher’s exact test and the Mann–Whitney test, respectively. The degree of agreement among reviewers for evaluating image quality was assessed using intraclass correlation coefficients (ICC) and their 95% CI, using a 2-way mixed, single measure, consistency model. ICC was interpreted according to Landis J. R. interpretation scale   complied to the eligibility criteria, out of which 46 did not consent to participate in the study. Sixty-one patients, 36 (59.1%) female and 25 (40.9%) males were prospectively enrolled to the study. The median age was 66 years (33-84 years), median weight was 67 kg (40-100 kg) and the tumor sub-types investigated were ten. Patients were injected with a median dose of 131 MBq (99-199 MBq) of 18F-FDG; 66% of the median standard institutional dose of 196 MBq (148-296 MBq). Twenty examinations were performed with PET scanner 1, 21 with PET scanner 2 and 20 with PET scanner 3. The mean time interval between tracer injection and the second PET scan time point was 75 min for PET scanner 1 and 80 min for PET scanners 2 and 3. Data is presented in Table As shown in Fig. p=0.237). Figures Vice versa, image quality had a mean score of 4.62 ± 0.23 for the PET-processed dataset and 4.54 ± 0.20 for the PET-native dataset, a difference that was not statistically significant  and 0.921 (95% CI= 0.855-0.943) respectively.PET processed images and PET-native images were easily and properly identified with almost perfect accuracy by all the four readers. Namely, PET-processed images in 97.9% of cases, and the PET-native images in 97.2% of cases.According to the standard of reference, 183 lesions were detected in 46 examinations. In 15 examinations, no lesions were detected. The mean number of lesions detected by the four reviewers was 182.75 ± 3.1 for the PET-processed examinations and 181.75 ± 2.8 for the PET-native images. No reviewer reported false-positive lesions in the PET-processed examinations. Results are presented in Table A mean number of 2.98 ± 0.05 lesions per patient were detected by all reviewers in PET-processed images and 2.99 ± 0.05 in PET-native images. Results are presented in Table max value measured in the target lesions (lesion with highest SUVmax) was 9.8 ± 8.8 in the PET-processed dataset and 11.4 ± 9.8 in the PET-native dataset. The mean SUVmean value of the liver was 2.1 ± 0.7 in the PET-processed dataset and 1.9 ± 0.7 in the PET-native dataset. The difference was not statistically significant in either .The mean SUVThe analysis of the evaluation of image quality and lesion detectability was performed in three sub-cohorts, stratifying the overall population by the three PET scanner models. Both the quantitative and qualitative results did not produce any significant differences between the scanner models and are presented in Table The total number of F18-FDG examinations performed in the facility in 2019 and the respective cost of the ordered radiopharmaceutical were used to calculate the potential net savings from the introduction of SubtlePET™. The business model projected 25% savings instead of 33%, to account for a possible increase in the radiopharmaceutical price from the supplier due to the reduction of the total amount ordered. The annual cost of the use of SubtlePET™ agreed by both parties was 15-20% of the gross annual radiopharmaceutical savings.This is the first real-world study to assess the clinical use of AI for dose reduced 18F-FDG PET/CT examinations and methodology to define a pricing model.A single center study on the feasibility of 18F-FDG dose reduction in PET/MR examinations was recently published, in which imaging of reduced doses was simulated by reconstruction with different percentages of the original 20 patient’s data . The stumax value measured in the target lesions, and the mean SUVmean value of the liver. Therefore, the AI-processed, 33% dose reduced images were deemed to be “non-inferior” to native images.In our study, the decision to reduce the administered dose of 18F-FDG by 33% was based on the assumption that the prolonged duration of the examination for two consecutive PET scans, would be less likely to lead to patient movement artifacts and more likely to be proved non-inferior than the previously described simulations reducing activity by 50%. Although the reviewers could identify whether they were reviewing the PET-processed or PET-native datasets in almost 98% of the cases, the mean image quality score of the datasets was not significantly different and there was “almost-perfect” agreement among reviewers. There was no significant difference in the mean number of lesions detected by the four reviewers compared to the reference standard, with no reviewer reporting false-positive lesions in the PET-processed examinations. There were also no significant statistical differences in the mean number of lesions detected per patient, mean SUVIn addition, there were no significant differences in the subjective image quality of the datasets and number of lesions detected between the different PET scanners.Based upon the equivalence of processed and native images, a business case was defined in which the cost of the algorithm was determined to be between 15 and 20% the overall cost saving in 18F-FDG ordered. This commercial arrangement was agreed between both AI developer and service provider for the network, with differing AI cost per scan based upon local 18F-FDG pricing and examinations volume.Ideally, a direct comparison of dose-reduced/processed images would be made to native images obtained from a scan performed according to the routine clinical protocol. A limitation of the study was that only one dose of 18F-FDG could be administered due to radiation protection limitations. This meant that the native images had to be acquired sequentially with a lower administered dose but longer acquisition times. A correction factor was used to account for this but may have affected the quality of the native images acquired therefore minimizing the differences between image quality. A further limitation was that the pathology was not controlled for, with ten different cancer types included in the 61-patient cohort. Although different pathological entities have differing avidities to 18F-FDG, the methodology chosen should have controlled for this variability. Although the reviewers were blinded, they were still able to accurately determine whether the scans were processed or native. This presents a potential bias, should the reviewers decide to assign a more favorable or worse image quality to the images they know are processed, depending on their own prejudice regarding artificial intelligence.TM in a reproducible manner. Further studies should be undertaken to determine the lower limit of 18F-FDG administration that AI processing will allow while still preserving diagnostic image quality, and whether these results are reproducible in pediatric population in which dose reduction is even more pertinent.To our knowledge, this study provides the first “real world” data comparing actual dose-reduced, AI processed images with native scans, rather than simulation. Given the robust methodology involving a large patient cohort, multiple blinded reviewers, and three different systems, we can be confident that for adult patients undergoing 18F-FDG PET, reducing the dose to 66% of standard produces images with non-inferior image quality when processed by SubtlePETAdditional file 1: Appendix 1. Seven PET-negative patients outside the study protocol were injected with standard 18F-FDG dose and acquired at 60 minutes post-injection. A subsequent second scan of the upper abdomen was obtained, using the same elapsed time used in the study protocol. By the comparison of the liver 18F-FDG uptake in the two scans, a correction factor of 1.063 was extracted and applied when comparing SUV values in PET-processed vs. PET-native images. Results are presented in Supplemental Table 1. Supplemental Table 1. Data for SUV correction factor. Supplemental Table 2. Study population.


Mycobacterium tuberculosis (M.tb) infection in the context of gene expression is still a scientific and diagnostic problem. The study was aimed to assess IL-18, IL-18 binding protein (IL-18BP), IL-18R, IFN-γ, and IL-37 mRNA expression in patients with active tuberculosis (ATB) and healthy volunteers with latent M.tb-infection (LTB) or M.tb-uninfected healthy controls (Control). The relative mRNA expression was assessed in the buffy coat blood fraction using the qPCR method. In total, 97 BCG-vaccinated Polish adults were enrolled in the study. The relative expression of IL-18 and IL-18BP mRNA was significantly elevated in the ATB and LTB groups. In ATB, but not LTB individuals, the overexpression of IL-18 and IL-18BP, as well as a significant increase in IFN-γ mRNA expression, might be considered as a manifestation of active tuberculosis disease. No statistically significant differences were observed in the IL-37 mRNA expression among the studied groups. Particularly noteworthy is the outstanding reduction in the relative expression of IL-18R mRNA in the LTB group as compared to the ATB and Control group. Reduced expression of IL-18R in LTB group may, at least partially, prevent the development of a pathological inflammatory reaction and promote the maintenance of homeostatic conditions between host immunity and M.tb.A thorough understanding of the processes modulating the innate and acquired immune response to  Mycobacterium tuberculosis (M.tb). TB affects about 10-million people in the world and is a cause of two-million deaths annually, according to the estimates of the World Health Organization [M.tb infection. These individuals have developed an efficient immune response that allows them to block the metabolic activity of the pathogen, but it does not provide its eradication. People with a latent TB infection (LTB) represent a reservoir of potential progress to disease, because about 5–10% of them will develop active TB disease, if not treated.Tuberculosis (TB) is the leading global cause of death from an infectious agent, M.tb infection is modulated by mRNA expression, which results in the production of cytokines and other proteins activating numerous cell populations. Among these, interleukin (IL)-18plays an important role—it induces NK cell cytotoxic activity and promotes the development of Th1 cell response. This mechanism is associated with the production of interferon (IFN)-γ, which is a key element in anti-mycobacterial protection. IL-18 was first described in 1989 as an “IFN-γ inducing factor” [M.tb was found in animals that were not able to produce IL-18 [The antigen-specific, as well as non-specific, response of the immune cells to  factor” ,3,4. Simce IL-18 . Moreovece IL-18 . In humace IL-18 .The IL-18 levels are also regulated by soluble IL-18 binding protein (IL-18BP), which is a natural inhibitor of IL-18. Under physiological conditions, the concentration of plasma IL-18BP is ~20 times higher than that of IL-18, which prevents IL-18 from binding to its cellular receptor. The gene coding for IL-18BP is located on chromosome 11 at position 11q13.4. The mRNA promoter region contains two response elements (RE), regulatory sequences sensitive to IFN-γ attachment, which results in increased gene expression and protein production .Cell activation by IL-18 occurs via the IL-18R receptor, which belongs to the IL-1 family, the members of which show structural and functional similarity. IL-18R is expressed on many cells, such as macrophages, NK cells, neutrophils, epithelial cells, and smooth muscle. The IL-18R receptor is a heterodimer that is composed of two polypeptide chains: IL-18Rα and IL-18Rβ. The IL-18Rα chain is responsible for ligand binding. However, it binds to IL-18 with low affinity. On the other hand, the IL-18Rβ chain functions as a co-receptor, which enhances the strength with which the receptor binds IL-18 and transmits the signal to the inside of the cell ,11,12. TA new member of the IL-1 family, IL-37, has gained increasing attention in recent years. IL-37, to which IL-18BP also has a high affinity, was found to be an important regulator of inflammation ,15. IL-3M.tb infection. This cytokine is mainly produced by activated CD4 (+) Th1 T lymphocytes, and it also has a key role in inducing nitric oxide (NO)-dependent apoptosis of mycobacteria infected macrophages. IFN-γ was shown to increase the expression of major histocompatibility complex (MHC) class I and II surface molecules and promote differentiation towards Th1 response. Deleterious mutations in the gene encoding the IFN-γ receptor predispose to the acute course of mycobacterial infection [M.tb infection status [M.tb infection (LTB), and healthy uninfected controls (Control).IFN-γ is a cytokine that is known to be crucial in regulating the immune response to nfection ,18.The hnfection . The expn status ,21. ThisM. bovis BCG in childhood, including 51 patients with active pulmonary ATB  aged 23–80 years, hospitalized and diagnosed in the Regional Center Hospital for Tuberculosis, Lung Diseases, and Rehabilitation in Lodz, Poland, 24 healthy volunteers, eight males, 16 females, with LTB (25–65 years old), and 22 healthy uninfected Controls , aged 18–66 years . Informed consent to use blood for research purposes was signed by all participants.A study group consisted of 97 adults that were vaccinated with 66 years . Active ® RNA Blood Mini set. Genetic material was isolated from 3.5 mL of peripheral blood obtained from all volunteers using EDTA tubes and the BD Vacutainer® Blood Collection system. The isolation process was fully compliant with the manufacturer’s guidelines. The isolation process was extended by an additional purification step using the RNase-Free DNase Set to obtain the purest product free of any genomic DNA. All of the procedures were carried out within no more than 2 h from the collection of a blood sample. Part of extracted RNA was used to visualize a product and obtain cDNA immediately after the isolation process; the rest of the genetic material was stored at −80 °C until analyzed.RNA isolation from the buffy coat obtained after centrifugation  was performed by the use of a commercial QIAampAt the end of the isolation procedure, 1.5 μL of each sample was pipetted into a sterile Eppendorf tube, which was then placed in an ice block, in order to assess the quality and quantity of RNA obtained. An additional water-containing blank was prepared to calibrate the device (NanoDrop), which was used for RNA elution in the final isolation step. For visualization of RNA, 1.2% agarose gel was prepared based on TAE buffer. Agarose gel was enriched after cooling with 10 μL of 5 mg/mL ethidium bromide. Each sample of RNA in a volume of 3.5 μL was heated to 70 °C in a water bath for 1 min. and then cooled on ice for another minute. An equal amount of loading buffer was then added to each of the samples, and the mixture was then loaded to the gel. The GeneRuler Plus DNA Ladder size 100 bp from ThermoFisher was used as the size standard. Electrophoresis was carried out at 90 V for 60 min. Subsequently, to visualize the obtained product, the gel was transferred to a Gel-Doc 2000 apparatus that was connected to a computer with Quantity One software. The analysis of the obtained gel allowed a clear distinction between the two isolated RNA fractions: 18S RNA and 28S RNA.cDNA was synthesized according to the manufacturer’s instructions of the iScript™ cDNA Synthesis Kit (Bio-Rad). In the first stage of the reverse transcription reaction, 1 μg matrix RNA previously tested for quality and integrity was transferred from an isolated sample to 0.2 μL Eppendorf tubes, followed by the addition of reagents that are necessary for the cDNA synthesis process according to the manufacturer’s proportions, which results in a mixture with a final volume of 20 μL. The reverse transcription reaction was carried out in a Biometra UNO II thermal cycler under conditions following the manufacturer’s guidelines. The resulting cDNA was stored at −20 °C until analyzed.qPCR was performed in a CFX96 Real-Time PCR Detection System (Bio-Rad). The reaction mixture (10 μL) contained 5 μL of iTaq universal SYBR Green Supermix, 0.5 μL of each primer (10 μM), 1 μL of cDNA, and 3 μL of nuclease-free water. Amplifications were performed using the following cycling profile: an initial activation step (95 °C for 3 min.) followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at a temperature appropriate for selected starters  for 10 sAnalysis of gene expression was done through a comparative method (ΔΔCt) in order to determine the relative level of expression of selected mRNAs. This method is based on calculating the differences in the level of expression of the test gene and the reference gene. The calculations use the threshold cycle (Ct) values of the qPCR reaction. Ct values were determined for both the test and reference genes in both the test and control samples, for which the differences between the individual Ct values (ΔCt) were then calculated.p-value < 0.05 was considered to be statistically significant. Statistical analyses were done using MedCalc  and GraphPad Prism 8  software.The expression of genes between the study groups was compared using Kruskal–Wallis’ non-parametric diagnostic test. A We had chosen to use the Reference Gene qPCR Panel BioRad with primers being designed and coated on the plate in lyophilized form for selected reference genes adequate for the analyzed material obtained from volunteers . The houMelt peak analysis demonstrated a single homogenous peak for all primer sets, including selected reference genes .M.tb infection (p = 0.023). A similar increase in the relative IL-18 expression level was observed among ATB patients; however, the difference in values for the group ATB and group Control group was not significant (p = 0.082)  A.p < 0.001) and healthy LTB individuals (p = 0.006) than in Control group volunteers (The relative level of IL-18BP mRNA expression was significantly higher in ATB patients (lunteers B.p < 0.001) and individuals from the Control group (p < 0.001)  C.p = 0.002) and LTB individuals (p = 0.029) than in the Control group , the enzyme of tryptophan metabolism, which leads to the formation of tryptophan metabolites, including quinolinic and picolinic acids [M.tb pathogenesis [M.tb-infected mice, IFN-γ receptor-deficiency in nonhaematopoietic cells led to a lack of IDO-1 expression and it was associated with exuberant neutrophil recruitment and increased mortality [M.tb. The IDO levels were elevated at the time of TB diagnosis and declined after TB treatment, which serves as evidence that IDO expression might be both: a useful diagnostic marker of active TB as well as prognostic factor in TB treatment of HIV-negative patients [Among many possible mediators of host response to ic acids . In animogenesis ,30,33,34ogenesis . In M.tbortality . Almeidaortality . The autpatients . Additiopatients , while Spatients . The incpatients ,37,39. IM.tb and host-immunity in latent tuberculosis infection. As an obligate intracellular pathogen, M.tb has numerous adaptive mechanisms of modifying cellular processes in the fight against the host immune response. In latent TB infection, M.tb bacilli benefit from epigenetic changes that occurred in the host immune system under mycobacterial infection [M.tb favorable environment in the host cells and promote mycobacterial survival, growth, and latency. In a study that was conducted among Chinese patients with pulmonary TB and healthy controls, single nucleotide polymorphisms in the IL-18R promoter were associated with genotype-specific methylation status and genotype-specific IL-18R expression [M.tb induces IL-37 production via enhancing TLR2 expression in human type II alveolar epithelial cells; this process might contribute to the persistence of M.tb infection [Listeria monocytogenes infection due to reduced production of colony-stimulating factors and increased macrophage apoptosis [M.tb infection [The presented results entitle us to hypothesize that: the increase in IL-18 gene expression, the lack of increase in IFN-γ gene expression, and the remarkably reduced expression of IL-18R gene may be a novel set of conditions that partially describe the homeostasis between nfection . These cpression . In the pression . Mannosenfection . Zhao etpoptosis . In our nfection . HoweverM.tb infection. In our future studies, we plan to test the expression of other genes that are tightly co-regulated with the IL-18 pathway.The role of IL-18, its binding protein IL-18BP, and IFN-γ in the development of the immune response against mycobacteria was confirmed by observing the increased level of the expression of these genes in the group of patients with active pulmonary TB. The reduced expression of IL-18R gene in healthy individuals with latent TB infection can, at least partially, prevent the development of a pathological inflammatory reaction and promote the maintenance of homeostatic conditions between host immunity and